Isolation of two distinct prion strains from a scrapie-affected sheep

We performed a transmission study using mice to clarify the characteristics of the most recent case of scrapie in Japan. The mice that were inoculated with the brain homogenate from a scrapie-affected sheep developed progressive neurological disease, and one of the scrapie-affected mice showed unique clinical signs during primary transmission. This mouse developed obesity, polydipsia, and polyuria. In contrast, the other affected mice exhibited weight loss and hypokinesia. In subsequent passages, the mice showed distinct characteristic scrapie phenotypes. This finding may prove that different prion strains coexist in a naturally affected sheep with scrapie

Isolation of Proteinase K-Sensitive Prions Using Pronase E and Phosphotungstic Acid

Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrPSc using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrPC, while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrPC

A short purification process for quantitative isolation of PrP(Sc) from naturally occurring and experimental transmissible spongiform encephalopathies

BACKGROUND: Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrP(C)) into a heat- and protease-resistant abnormal isoform (PrP(Sc)). Detection of PrP(Sc) in individuals is widely utilized for the diagnosis of prion diseases. METHODS: TSE brain tissue samples have been processed in order to quantitatively isolate PrP(Sc). The protocol includes an initial homogenization, digestion with proteinase K and salt precipitation. RESULTS: Here we show that over 97 percent of the PrP(Sc) present can be precipitated from infected brain material using this simple salting-out procedure for proteins. No chemically harsh conditions are used during the process in order to conserve the native quality of the isolated protein. CONCLUSION: The resulting PrP(Sc)-enriched preparation should provide a suitable substrate for analyzing the structure of the prion agent and for scavenging for other molecules with which it may associate. In comparison with most methods that exist today, the one described in this study is rapid, cost-effective and does not demand expensive laboratory equipment

A C-Terminal Protease-Resistant Prion Fragment Distinguishes Ovine “CH1641-Like” Scrapie from Bovine Classical and L-Type BSE in Ovine Transgenic Mice

The protease-resistant prion protein (PrPres) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrPres in four “CH1641-like” natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if “CH1641-like” isolates might be linked to L-type BSE. We found less diglycosylated PrPres than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrPres form (PrPres #1) to L-type BSE. However, the “CH1641-like” isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrPres product (PrPres #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrPres #1 and PrPres #2 resulted in enrichment in PrPres #2, and demonstrated the presence of mono- and diglycosylated PrPres products. PrPres #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrPres #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between “CH1641-like” ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model

Emergence of Classical BSE Strain Properties during Serial Passages of H-BSE in Wild-Type Mice

BACKGROUND: Two distinct forms of atypical spongiform encephalopathies (H-BSE and L-BSE) have recently been identified in cattle. Transmission studies in several wild-type or transgenic mouse models showed that these forms were associated with two distinct major strains of infectious agents, which also differed from the unique strain that had been isolated from cases of classical BSE during the food-borne epizootic disease. METHODOLOGY/PRINCIPAL FINDINGS: H-BSE was monitored during three serial passages in C57BL/6 mice. On second passage, most of the inoculated mice showed molecular features of the abnormal prion protein (PrP(d)) and brain lesions similar to those observed at first passage, but clearly distinct from those of classical BSE in this mouse model. These features were similarly maintained during a third passage. However, on second passage, some of the mice exhibited distinctly different molecular and lesion characteristics, reminiscent of classical BSE in C57Bl/6 mice. These similarities were confirmed on third passage from such mice, for which the same survival time was also observed as with classical BSE adapted to C57Bl/6 mice. Lymphotropism was rarely detected in mice with H-BSE features. In contrast, PrP(d) was detectable, on third passage, in the spleens of most mice exhibiting classical BSE features, the pattern being indistinguishable from that found in C57Bl/6 mice infected with classical BSE. CONCLUSION/SIGNIFICANCE: Our data demonstrate the emergence of a prion strain with features similar to classical BSE during serial passages of H-BSE in wild-type mice. Such findings might help to explain the origin of the classical BSE epizootic disease, which could have originated from a putatively sporadic form of BSE

Regulating Factors of PrPres Glycosylation in Creutzfeldt-Jakob Disease - Implications for the Dissemination and the Diagnosis of Human Prion Strains

OBJECTIVE: The glycoprofile of pathological prion protein (PrP(res)) is widely used as a diagnosis marker in Creutzfeldt-Jakob disease (CJD) and is thought to vary in a strain-specific manner. However, that the same glycoprofile of PrP(res) always accumulates in the whole brain of one individual has been questioned. We aimed to determine whether and how PrP(res) glycosylation is regulated in the brain of patients with sporadic and variant Creutzfeldt-Jakob disease. METHODS: PrP(res) glycoprofiles in four brain regions from 134 patients with sporadic or variant CJD were analyzed as a function of the genotype at codon 129 of PRNP and the Western blot type of PrP(res). RESULTS: The regional distribution of PrP(res) glycoforms within one individual was heterogeneous in sporadic but not in variant CJD. PrP(res) glycoforms ratio significantly correlated with the genotype at codon 129 of the prion protein gene and the Western blot type of PrP(res) in a region-specific manner. In some cases of sCJD, the glycoprofile of thalamic PrP(res) was undistinguishable from that observed in variant CJD. INTERPRETATION: Regulations leading to variations of PrP(res) pattern between brain regions in sCJD patients, involving host genotype and Western blot type of PrP(res) may contribute to the specific brain targeting of prion strains and have direct implications for the diagnosis of the different forms of CJD

Propagation of RML Prions in Mice Expressing PrP Devoid of GPI Anchor Leads to Formation of a Novel, Stable Prion Strain

PrPC, a host protein which in prion-infected animals is converted to PrPSc, is linked to the cell membrane by a GPI anchor. Mice expressing PrPC without GPI anchor (tgGPI- mice), are susceptible to prion infection but accumulate anchorless PrPSc extra-, rather than intracellularly. We investigated whether tgGPI− mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrPSc. We found that RML and ME7, but not 22L prions propagated in tgGPI− brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrPSc (PrPres) in RML- or ME7-infected tgGPI− brain were 25–50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrPSc and SFL PrPSc are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice

A New Method for the Characterization of Strain-Specific Conformational Stability of Protease-Sensitive and Protease-Resistant PrPSc

Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep

Defining the Conformational Features of Anchorless, Poorly Neuroinvasive Prions

Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same infectious prion develop fibrillar or nonfibrillar aggregates, respectively, and show a striking divergence in the disease pathogenesis. To better understand how a prion's physical properties govern the pathogenesis, infectious anchorless prions were passaged in mice expressing anchorless prion protein and the resulting prions were biochemically characterized. Serial passage of anchorless prions led to a significant decrease in the incubation period to terminal disease and altered the biochemical properties, consistent with a transmission barrier effect. After an intraperitoneal exposure, anchorless prions were only weakly neuroinvasive, as prion plaques rarely occurred in the brain yet were abundant in extracerebral sites such as heart and adipose tissue. Anchorless prions consistently showed very high stability in chaotropes or when heated in SDS, and were highly resistant to enzyme digestion. Consistent with the results in mice, anchorless prions from a human patient were also highly stable in chaotropes. These findings reveal that anchorless prions consist of fibrillar and highly stable conformers. The additional finding from our group and others that both anchorless and anchored prion fibrils are poorly neuroinvasive strengthens the hypothesis that a fibrillar prion structure impedes efficient CNS invasion